The present invention relates to a prognostic method and prognostic means based on polymorphisms in the vitamin D receptor and collagen Ixcex11 genes. In particular, the present invention relates to a method for determining susceptibility to bone damage by screening for polymorphisms in vitamin D receptor or collagen Ixcex11 genes.
Osteoporosis is a common disease characterized by reduced bone mineral density (BMD), deterioration of bone micro-architecture and increased risk of bone damage, such as fracture.1 It is a major public health problem which affects quality of life and increases costs to health care providers. In European populations, one in three women and one in twelve men over the age of fifty is at risk. The disease effects 25 million people in the USA, where the incidence of disease is 25% higher than it is in the UK, and a further 50 million people in Japan and Europe combined. It is estimated that by the middle of the next century the number of osteoporosis sufferers will double in the West, but may increase six-fold in Asia and South-America. Fracture is the most serious endpoint of osteoporosis, particularly fracture of the hip which affects up to 1.7 million people worldwide each year. It is estimated that by the year 2050, the number of hip fractures worldwide will increase to over 6 million, as life expectancy and age of the population increase.
Treatment of osteoporosis is unsatisfactory. In particular, once bone damage has occurred as a result of osteoporosis, there is little a physician can do other than let the bone heal. In the elderly, this may be a slow and painful process. Diagnosis of those at risk of developing osteoporosis allows more effective preventative measures. Strategies for the prevention of this disease include development of bone density in early adulthood, and minimisation of bone loss in later life. Changes in lifestyle, nutrition and hormonal factors have been shown to affect bone loss.7-14 
Osteoporosis can be considered a complex genetic trait with variants of several genes underlying the genetic determination of the variability of the phenotype. Low bone mineral density (BMD) is an important risk factor for fractures, the clinically most relevant feature of osteoporosis. Segregation analysis in families has shown that BMD is under polygenic control12,13 while, in addition, biochemical markers of bone turnover have also been shown to have strong genetic components14-16. Several candidate genes have been analysed in relation to BMD but the most widely studied gene in this respect, the vitamin D receptor (VDR) gene, explains only a small part of the genetic effect on BMD8. Numerous studies, focussing on the BsmI allele of the vitamin D receptor gene have concluded that absence of the restriction site correlates with low bone mineral density. Most genetic analyses have focussed on BMD as a determinant of fracture risk and not so much on fractures themselves as an endpoint in the analysis. Recently, an SpI polymorphism in the COLIA1 gene encoding the most abundant bone matrix protein, was found to be associated with reduced BMD and, more importantly, also with increased risk of osteoporotic fracture9,10. An emerging theme from these studies seems to be the association of the absence of the BsmI restriction site with reduced bone mineral density, which in turn signifies increased risk of bone damage such as fracture.
Few studies have addressed genetic association with the clinically most important endpoint of osteoporosis, namely bone damage, in particular fracture. Accordingly, it is an object of the present invention to improve the prognosis of predisposition or susceptibility to bone damage.
In a first aspect of the present invention, there is provided a method of determining susceptibility to bone damage in a subject, said method comprising analyzing genetic material of a subject to determine which allele(s) of the vitamin D receptor gene is/are present.
Thus, the present invention satisfies the pressing need for identification of those individuals susceptible to bone damage, thus facilitating the development of preventative measures. For example, those at risk may avoid damage by modifying their lifestyle and implementing bone strengthening measures, such as regular exercise and a healthy diet. Typically, the method of the present invention comprises analysis of polymorphisms in the VDR gene to determine susceptibility to bone damage. The method may include determining whether one or more particular alleles are present, or which combination of those alleles (i.e. the haplotype) is present. The method may further comprise determining whether subjects are homozygous or heterozygous for alleles or haplotypes of the vitamin D receptor gene.
Vitamin D is a potent regulator of bone and calcium homeostasis, as well as of cellular differentiation and replication in many tissues, and mediates its effects through the vitamin D receptor (VDR). Cloning of the vitamin D receptor has shown it to be a member of the ligand-activated superfamily, which are natural regulators of a number of physiological and developmental processes. Evidence suggests that the vitamin D receptor activates expression of the osteocalcin gene through interaction with a palindromic sequence in the promoter of the gene.27 The osteocalcin gene product is a marker of bone turnover in normal and disease states, and inter-individual variation in its circulating levels have been associated with polymorphisms in the vitamin D receptor gene.
The vitamin D receptor gene (12q12) comprises inherited polymorphisms between exon 7 and the 3xe2x80x2 UTR of the VDR gene, as shown in FIG. 1. These alleles are denoted B/b, A/a and T/t for restriction enzyme sites BsmI, ApaI and TaqI respectively (or enzymatic or chemical procedures having similar specificity), where a lower case letter denotes the presence of a wild type restriction site which is capable of being cleaved, and a capital letter denotes the presence of a mutant restriction enzyme site which is not capable of being cleaved by the relevant restriction enzyme. For the purposes of the present invention, determination of which alleles are present in a particular gene may be referred to as determining the genotype of a subject for a particular gene. It is apparent from the above that each copy of the vitamin D receptor gene will comprise a specific combination of the three alleles, this combination being referred to as the haplotype of the gene. For example, the haplotype may be baT, indicating the presence of cleavable BsmI and ApaI sites, and a non-cleavable TaqI site. Direct haplotyping of the VDR gene has allowed five different haplotypes to be determined, of which three are common.11 
The present invention is based upon the surprising observation of a correlation between the presence of the b allele of the vitamin D receptor and susceptibility to/or risk of (where the terms are used interchangeably) bone damage, such as fracture. The invention goes further to show that presence of the a and/or T alleles, and in particular the haplotype baT is/are associated with increased risk of bone damage. A subject having the baT haplotype may show a higher risk of fracture compared to a subject having the bAt, or BAt haplotype which confers the lowest risk of fracture. The results are unexpected, as previous studies have shown the b allele, and particularly the baT haplotype, to be associated with high bone density, which itself is not associated with fracture risk. Thus, in contrast to previous results, the present inventions have shown that susceptibility to bone damage is independent of bone mineral density in a subject. By screening for the presence of alleles of the vitamin D receptor gene, susceptibility to bone damage may be assessed without the need for analysis of bone mineral density.
Preferably, the method of the first aspect of the present invention further comprises determining whether the alleles present are associated with risk of bone damage. This may be performed by comparing the alleles present in a subject with those known to be associated with risk of bone damage. For example, a visual aid detailing alleles and the relative risk of bone damage associated therewith may be used to determine whether the genotype of the subject is associated with a high or low risk of bone damage.
The first aspect of the present invention may also comprise the additional step of determining aspects of calcium metabolism, such as calcium levels in a subject. Preferably, the daily calcium intake is measured. This feature of the first aspect is based on the observation that the correlation between vitamin D receptor genotype and bone damage may be dependent upon dietary calcium intake. Specifically, in subjects having low calcium intake, genotype dependent risks may be greater.
The method of the present invention may be performed in vitro. Preferably, the method is performed on tissue or fluid removed from the body of the subject. Thus, the present invention relates to a non-invasive diagnostic method, the results of which provide an indication of susceptibility to bone damage but do not lead to a diagnosis upon which an immediate medical decision regarding treatment has to be made.
The present invention may be performed on any subject for whom it is desirable to determine risk of bone damage. Preferably, the subject may be a mammal. Most preferably, the subject is a human, preferably a female.
Bone damage may be any form of structural damage including fractures, breaks, or chips. The term may also include biological degradation or deterioration of bone. Typically, the term bone damage does not include low bone density. This is in line with the finding that risk of bone damage is independent of bone density. Fracture may be defined as the clinically most important endpoint, and thus the method of the first aspect of the invention preferably relates to a method of determining risk of fracture. Although such bone damage will usually be the result of osteoporosis, it is irrelevant for the purposes of the present invention whether a subject has first been diagnosed as having osteoporosis.
In a preferred feature of the first aspect of the present invention, there is provided a method of determining susceptibility to bone damage, said method comprising analysing the genetic material of a subject to determine which of the B/b, Ala or T/t alleles of the vitamin D receptor are present. The method may comprise determining whether more than one of the above alleles is present. The subject may be further classified as heterozygous or homozygous for one or more of these alleles. Preferably, the method comprises the additional step of determining whether the allele(s) present are associated with risk of bone damage, wherein presence of the b or a alleles is associated with increased risk of bone damage, and presence of the t allele is associated with reduced risk of bone damage. Homozygosity for the a or b allele may further increase the susceptibility to bone damage in a subject, while homozygosity for the t allele may further decrease susceptibility.
In a preferred feature of the first aspect of the present invention, there is provided a method of determining susceptibility to bone damage in a subject, said method comprising analysing the genetic material of a subject to determine the haplotype of the BsmI, ApaI and TaqI alleles at the vitamin D receptor. Preferably, said method comprises determining whether the haplotype of the subject is associated with risk of bone damage, wherein the haplotype baT is associated with high risk of bone damage. A subject homozygous for said haplotype may be at a higher risk of bone damage than those heterozygous for the haplotype.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to bone damage, said method further comprising analysing the genetic material of a subject to determine which allele of the collagen Ixcex11 (17q22) gene is present. An allele in this gene may be denoted by S/s, where s indicates the presence of a G to T polymorphism at the SpI restriction site. This feature of the present invention is based on the observation that the correlation between increased fracture risk and VDR genotype may be collagen Ixcex11 genotype dependent. Specifically, in those subjects having vitamin D receptor alleles indicating risk of bone damage, the risk may be increased in those subjects also carrying a G to T polymorphism at the SpI site of the collagen Ixcex11 gene.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to bone damage, said method comprising determining the copy number of the B/b, A/a or T/t alleles or haplotype of the vitamin D receptor gene and/or the S/s alleles of the collagen Ixcex11 gene, where an increase in copy number is associated with increased risk of bone damage.
The present invention may be performed using any suitable method known in the art. Preferably, a tissue or fluid sample is first removed from a subject. Examples of suitable samples include blood, mouth or cheek cells, and hair samples containing roots. Other suitable samples would be known to the person skilled in the art. The genetic material is then extracted from the sample, using any suitable method. The agenetic material may be DNA or RNA, although preferably DNA is used. For example, the DNA may be extracted using the technique described in Sambrook et al (Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory Press). Determination of the genotype of a subject may then be carried out using the extracted DNA, employing any one of the following techniques:
Southern blot analysis following digestion with one or more appropriate restriction enzymes.
PCR amplification followed by digestion with one or more appropriate restriction enzymes and, optionally, separation of digestion products by gel electrophoresis.
Sequencing of a relevant gene fragment by any suitable method.
Visualization of heteroduplex patterns, for example on PAA or agarose gels, where different patterns may indicate the presence of one or more specific alleles.
Separation of DNA fragments using denaturing gradient gels, wherein the degree of separation will depend upon the presence or absence of one or more polymorphic restriction sites.
Separation using SSCP analysis, the patterns of which will depend upon the presence or absence of one or more polymorphic restriction sites.
Use of allele specific oligonucleotides, hybridization patterns of which will be specific for various combinations of alleles.
Methods such as OLA, Taqman or dot-blot for the detection of known mutations.
Visualization of DNA sites using fluorescent labelled probes for alleles of interest.
RFLP analysis.
Where it is desirable to use particular restriction enzymes in performing the present invention, the skilled person will understand that enzymatic or chemical procedures having similar specificities may also be used. For example, restriction enzymes having similar specificity (isoschizomers) to those described herein may be used, or chemical degredation procedures with DNA or RNA cutting specificity.
Other techniques suitable for determining the genotype of a subject may be used in the present invention.
Where the haplotype of a gene is to be determined, it is preferable to use a direct haplotyping method, as described in Uitterlinden et al11. In such a method, the relevant portion of the gene is amplified and then subjected to restriction enzyme digestion, in order to determine the presence or absence of restriction enzyme sites. Thus, for example, where the haplotype of the vitamin D receptor gene is to be determined, the portion of the gene between exon 7 and the 3xe2x80x2 UTR may be amplified, and the amplified DNA digested with the BsmI, ApaI or TaqI restriction enzymes. Gel analysis may then be used to determine which alleles are present.
Preferably, a fragment may be amplified using polymerase chain reaction (PCR) techniques, to produce copies which, where the fragment is of the vitamin D receptor, are at least 1000 base pairs in length, and most preferably at least 1800 base pairs in length. Where the fragment to be amplified is of the collagen Ixcex11 gene, PCR primers may be selected to amplify a fragment which is at least 50 base pairs in length, preferably at least 200 base pairs in length. PCR techniques are well known in the art, and it is within the ambit of the skilled person to identify primers for amplification of the appropriate region of the above genes, namely the region from exon 7 to the 3xe2x80x2 UTR of the vitamin D receptor gene and the first intron of the collagen Ixcex11 gene. PCR techniques are described in EP-A-0200362 and EP-A-0201184.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to bone damage in a subject, said method comprising amplifying a fragment comprising a portion of the region from exon 7 to the 3xe2x80x2 UTR of the vitamin D receptor gene, and determining which allele(s) in the vitamin D receptor is/are present. Primers suitable for amplification of said portion of the vitamin D receptor gene would be readily available to a person skilled in the art Examples of such primers include:
1. 5xe2x80x2-CAACCAAGACTACAAGTACCGCGTCAGTGA-3xe2x80x2 (SEQ ID NO:1) and/or
2. 5xe2x80x2-GCAACTCCTCATGGCTGAGGTCTC-3xe2x80x2 (SEQ ID NO:2)
In features of the present invention where it is desirable to determine which allele of the collagen Ixcex11 gene is present, at least a portion of the first intron of the collagen Ixcex11 gene may be amplified, followed by determination of the presence of a SpI restriction site. Suitable primers include:
1. 5xe2x80x2-TAACTTCTGGACTATTTGCGGACTTTTTGG-3xe2x80x2 (SEQ ID NO:3) and/or
2. 5xe2x80x2-GTCCAGCCCTCATCCTGGCC-3xe2x80x2 (SEQ ID NO:4)
Additional primer sequences are described in Grant et al9.
Where the amplified portion of the gene is larger than the above defined portions of the genes containing the relevant alleles, it is preferable to avoid the inclusion of vitamin D receptor or collagen Ixcex11 gene sequences which comprise any one of the BsmI, ApaI or TaqI restriction sites of the vitamin D receptor gene, or the SpI site of the collagen Ixcex11 gene.
In a second aspect of the present invention, there is provided a method of therapy, said method comprising treating a subject diagnosed as being at risk of bone damage, to reduce the risk of bone damage. Preferably, the subject is diagnosed as being at risk of bone damage in accordance with the first aspect of the present invention.
Therapy may in the form of preventative or palliative care. Suitable treatments include modifications to lifestyle, regular exercise and changes in diet to strengthen bones, and hormone therapy. Suitable treatments, including pharmaceutical preparations to reduce bone loss, would be known to physicians and persons skilled in the art. Examples include anabolic steroids, bisphosphonates, vitamin D preparations, calcium supplements and Hormone Replacement Therapy.
In a third aspect of the present invention, there is provided a method of predicting the response of a subject to treatment, said method comprising analysing genetic material of a subject to determine which allele(s) of the vitamin D receptor gene and/or collagen Ixcex11 gene is/are present. Preferably, the method includes determining whether the subject is susceptible to bone damage. Where a subject has been determined as susceptible to bone damage, the method may further comprise administering the appropriate treatment. The effect of a therapeutic or preventative agent may depend on the underlying cause of the heart disease, and in some cases it may be preferable to avoid the use of certain treatments. This aspect of the present invention may also be useful for identifying agents which may be used in the treatment of bone damage.
In a fourth aspect of the present invention, there is provided use of a kit to determine which allele(s) of the vitamin D receptor gene and/or collagen Ixcex11 gene is/are present, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of the vitamin D receptor and/or collagen Ixcex11 genes, and (ii) means for determining which allele(s) is/are present in said genes.
Preferably, the primer molecules are suitable for amplification of at least a portion of the region between exon 7 and the 3xe2x80x2 UTR of the vitamin D receptor gene, and/or a portion of the first intron of the collagen Ixcex11 gene. Examples of suitable primers are described above.
Means for determining which allele(s) is/are present in the vitamin D receptor gene, and/or collagen Ixcex11 gene may include any reagents or molecules necessary for use in any of the methods described above. For example, where PCR followed by DNA digestion is used, said means preferably include PCR reagents and one or more of the BsmI, ApaI, TaqI or SpI restriction enzymes. Where the method employs Southern Blotting, heteroduplex visualization, or fluorescent labelling techniques for example, probes which bind to the appropriate regions of the vitamin D receptor gene, and/or collagen Ixcex11 gene may be included. Where necessary, such probes may be labelled to allow detection, for example by nick-translation, radio- or fluorescent-labelling, or random primer extension whereby the non-labelled nucleotides serve as a template for the synthesis of labelled molecules. Other methods of labelling probes are well known in the art.
In a preferred feature of the fourth aspect of the present invention, there is provided use of a kit further comprising means for indicating correlation between the genotype of a subject and risk of bone damage. Said means may be in the form of a chart or visual aid, which indicate that presence of the b allele or baT haplotype, and the S allele of the couagen Ixcex11 gene is associated with increased fracture risk.
In a fifth aspect of the present invention, there is provided a kit for determining risk of bone damage in a subject, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of the vitamin D receptor and/or collagen Ixcex11 genes; (ii) means for determining which allele(s) is/are present in said genes; and (iii) means for indicating correlation between the allele(s) and risk of bone damage.
In a preferred feature of the fifth aspect, the kit may also comprise DNA control samples, for comparison with DNA sequences of a subject. The control samples may comprise the sequence of one or more alleles of the vitamin D receptor or collagen Ixcex11 genes, or may comprise the sequence of various haplotypes.
Preferred features of each aspect of the present invention are as for each other aspect, mutatis mutandis.
The present invention will now be described in detail with reference to the following examples and figures.
FIG. 1 is a schematic presentation of the region between exon 7 and the 3xe2x80x2 UTR of the vitamin D receptor genes.